Biochemical and Immunological Characterization of - Journal of Virology

JOURNAL OF VIROLOGY, Jan. 1980, p. 196-207 0022-538X/80/91-0196/12$02.00/0

Vol. 33, No. 1

Biochemical and Immunological Characterization of Polyproteins Coded for by the McDonough, Gardner-Arnstein, and Snyder-Theilen Strains of Feline Sarcoma Virus MARIANO BARBACID,* ANNE V. LAUVER, AND SUSHILKUMAR G. DEVARE Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, Maryland 20205

The McDonough (SM), Gardner-Arnstein (GA), and Snyder-Theilen (ST) strains of feline sarcoma virus (FeSV) code for high-molecular-weight polyproteins that contain varying amounts of the amino-terminal region of the FeLV gag gene-coded precursor protein and a polypeptide(s) of an as yet undetermined nature. The SM-FeSV primary translational product is a 180,000-dalton polyprotein which is immediately processed into a highly unstable 60,000-dalton molecule containing the pl5-pl2-p30 fragment of the FeLV gag gene-coded precursor protein and a 120,000-dalton FeSV-specific polypeptide. The GA- and ST-FeSV genomes code for polyproteins of 95,000 and 85,000 daltons, respectively, which in addition to the amino-terminal moiety (pl5-p12 and a portion of p30) of the FeLV gag gene-coded precursor protein also contain FeSV-specific polypeptides. However, the GA- and ST-FeSV polyproteins appear to be relatively stable molecules (half-lives of around 16 h) and are not significantly processed into smaller polypeptides. Immunological and biochemical analysis of each of the above FeSV translational products revealed that the sarcoma-specific regions of the GA- and ST-FeSV polyproteins are antigenically cross-reactive and exhibit common methionine-containing peptides. These findings favor the concept that these sarcoma-specific polypeptides are coded for by the similar subsets of cellular sequences incorporated into the GA- and ST-FeSV genomes during the generation of these transforming agents.

Transforming viruses have been isolated from a number of avian and mammalian species. They appear to have originated by a mechanism involving recombination between infectious type C (helper) viruses and specific subsets of cellular genetic information (1, 2, 6, 8, 12, 15, 16, 20, 23, 30, 36, 37, 40, 43, 44). With the exception of Rous sarcoma virus, all transforming viruses known to date are replication defective, requiring a helper type C virus to propagate from cell to cell. Efforts to understand the mechanisms by which these defective viruses exert their oncogenic action have recently focused on the identification of gene products responsible for malignant trans-

formation. It has been well documented that the genomes of these transforming viruses contain sequences from the helper type C virus involved in their generation (1, 2, 6, 8, 12, 15, 23, 30, 32a, 33, 43, 44). Some isolates express sequences encompassing at least the 5' moiety of the helper gag gene (1-3, 6, 7, 9, 21, 24, 25, 29-31, 34). However, genetic experiments have revealed that expression of these helper structural proteins is not required for malignant transformation (1-3, 6, 30). More recently, certain transforming viruses have been found to code for polyproteins that

contain, in addition to the amino-terminal region of the gag gene translational product, a polypeptide(s) of an as yet undetermined nature. These isolates include the MC29 and MH2 strains of avian acute leukemia viruses (9, 24, 29), avian erythroblastosis virus (21), and several mammalian isolates such as Abelson murine leukemia virus (MuLV) (32, 45), the T-8 virus (34), the Friend strain of spleen focus-forming virus (7), and three independent isolates of feline sarcoma virus (FeSV) (25, 31, 35). In the present report we have prepared hightitered antibodies against both the helper and sarcoma virus-specific domains of polyproteins coded for by the McDonough (SM) (28), Gardner-Arnstein (GA) (18) and Snyder-Theilen (ST) (39) strains of FeSV. These antisera, along with tryptic fingerprinting analysis of each of these molecules, have allowed us to characterize these polyproteins and to establish their immunological and biochemical relationships. These results have implications concerning the origin of these sarcoma viruses and bear also on the

putative biological role of these polyproteins. MATERIALS AND METHODS Cells and viruses. Cells were grown in Dulbecco's 196

VOL. 33, 1980

modification of Eagle medium supplemented with 10% calf serum (Colorado Serum Co., Denver, Colo.). Mink lung MvlLu (ATCC 64) and normal rat kidney (NRK) (13) cells nonproductively transformed by SM-FeSV (28), clones 116A and 124, respectively; by GA-FeSV (18), clones 65-5 and 128-4, respectively; and by STFeSV (39), clones 61-1 and 52-7, respectively, have been reported elsewhere (31). FeSV (WM 292-A) viral pseudotypes were obtained by superinfection of the respective nonproducer MvlLu-transformed cell lines with amphotropic wild mouse (WM 292-A) type C virus (17). Finally, FeLV propagated in A673 human cells (29) was utilized for antibody absorption experiments.

Isolation of FeSV-coded polyproteins. A 10-mg amount of isopycnically purified SM-, GA-, or STFeSV (WM 292-A) pseudotypes was pelleted (100,000 x g for 2 h) and resuspended in 1 ml of a 50 mM Trishydrochloride buffer (pH 8.0) containing 8 M guanidine-hydrochloride, 50 mM dithiothreitol, 1 mM EDTA, 1% Triton X-100, and 10% sucrose (wt/vol). After adding appropriate molecular weight markers, the disrupted virus preparation was applied on top of an agarose (A5m, BioRad) column (1.5 by 90 cm) equilibrated with a 20 mM sodium phosphate buffer, pH 6.5, containing 6 M guanidine-hydrochloride and 10 mM dithiothreitol (14). Fractions (0.8 ml) were collected and dialyzed against a 10 mM Tris-hydrochloride buffer (pH 7.8) containing 200 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, and 0.1 mM p-methylsulfonilfluoride. Dialyzed fractions were tested as competing antigens at twofold serial dilutions in homologous competition radioimmunoassays for FeLV p15 and p12 proteins as previously described (31, 32a). Fractions containing both FeLV p15 and p12 antigenic determinants were pooled, aliquoted, and utilized as immunogens. Antisera. Antisera elicited against FeSV-coded polyproteins were obtained by immunizing goats either with proteins purified as described above or with autologous cells infected with the corresponding FeSV (WM 292-A) pseudotype. Antisera directed against FeSV-specific antigenic determinants were obtained by absorbing the above antisera with disrupted FeLV bound to a Sepharose 4B matrix. Briefly, 10 mg of isopycnically purified FeLV was incubated with 1.5 g of CNBr-activated Sepharose 4B (Pharmacia) in 10 ml of 100 mM sodium borate buffer, pH 8.5, containing 0.5% of Triton X-100. After eliminating unbound proteins by sequential washing with 50 mM sodium acetate buffer, pH 4.0, and 100 mM Tris-hydrochloride buffer, pH 8.5, containing 1 M NaCl, the resin was extensively equilibrated with a 20 mM Tris-hydrochloride buffer, pH 7.8, containing 200 mM NaCl and 1 mM EDTA. The washed resin was incubated at room temperature with appropriate antiserum (3 to 5 ml) in a rotary shaker for 3 h and poured into a small plastic column (1 by 10 cm), and the eluant was collected. The degree of absorbtion of anti-FeLV antibodies was monitored both by immunoprecipitation of [3S]methionine-labeled FeLV-infected cell extracts and radioimmunoprecipitation of purified 1251-labeled FeLV proteins (see below). Caprine antisera directed against FeLV virions or

FeSV-CODED POLYPROTEINS

197

against purified proteins including FeLV p15, p30 and gp7O, and Rauscher-MuLV plO and reverse transcriptase were provided by R. Wilsnack (Huntingdon Research Center, Baltimore, Md.) through the Virus Cancer Program, National Cancer Institute. Radioimmunoassays. Double-antibody precipitation and competition radioimmunoassays involving the use of '25I-labeled FeLV p15, p12, p30, and gp7O (31, 41) and Rauscher-MuLV plO and reverse transcriptase (5, 26) proteins were performed as previously described (31). Inmunoprecipitation and electrophoretic analysis of labeled cell extracts. Subconfluent cul-

tures of FeSV-transformed nonproducer or control cells were washed with methionine-free Dulbecco-

modified Eagle medium and labeled for the required length of time (2 h, unless otherwise stated) with 3 ml of the same medium but containing 50 ,Ci of [35S]methionine per ml (specific activity, 1,200 Ci/mmol, New England Nuclear Corp.). Radioactive tissue culture fluids were removed and cells lysed with 2 ml of a 10 mM sodium phosphate buffer, pH 7.5, containing 100 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS) per petri dish. The cell lysate was clarified and preincu-

bated for 2 h in the cold with a 1:20 volume of normal goat serum and a 1:10 volume of freshly prepared, Formalin-fixed Staphylococcus aureus. The lysate was centrifugated for 30 min at 100,000 x g, and the pellet was discarded. The supernatant was divided into 0.15ml aliquots, incubated overnight in the cold with the appropriate antiserum dilution, and then mixed with 0.2 ml of protein A Sepharose 4B beads (Pharmacia). After incubation for 1 h, the antigen-antibody complexes bound to the protein A Sepharose beads were collected by centrifugation (2 min at 20,000 x g), washed three times with lysing buffer, and finally dissociated by incubation at 95°C for 2 min. Sepharose beads were removed by centrifugation, and the supernatant was applied to a 6 to 12% linear SDS-polyacrylamide gradient gel. Cells were processed for fluorography and exposed at -70°C to Kodak XR-5 film as previously described (10). Two-dimensional tryptic peptide analysis. FeSV-coded proteins labeled with [35S]methionine were identified on SDS-polyacrylamide gels by autoradiography. The gels were excised and the protein was eluted with 0.05 M ammonium bicarbonate buffer (pH 8.0) containing 0.1% SDS at 37°C overnight. The eluted protein was precipitated with 20% trichloroacetic acid along with 100 ug of bovine serum albumin at 4°C. The precipitate was washed with ice-cold 20% trichloroacetic acid three times and twice with acetone. Dry pellet was oxidized with performic acid (22) for 20 min at room temperature, diluted with water, and lyophilized. The samples were suspended in 0.05 M ammonium bicarbonate buffer (pH 8.0) and treated with tolylsulfonyl phenylalanyl chloromethyl ketonetrypsin (Worthington Biochemicals Corp., Freehold, N.J.) at enzyme-protein ratio 1:20 for 8 h at 37°C. Samples were lyophilized and were subjected to electrophoresis on cellulose-coated glass plates (10 by 10 cm, E. Merek Laboratories, Inc., Elmsford, N.J.) with acetic acid-pyridine-water (50:5:44.5, vol/vol) buffer, pH 3.5, for 1.5 h. The plates were chromatographed in

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J. VIROL.

second dimension with butanol-acetic acid-pyridinewater (32:5:25:5, vol/vol) as solvent. The plates were dried and coated with 0.4% PPO (2,5-diphenyloxazole) in 2-methylnaphthalene (11), and peptide maps were

were immunized with autologous cells infected with GA-FeSV (WM 292A) or ST-FeSV (WM 292A) virus pseudotypes. Antisera directed against FeSV-specific translational products visualized by fluorography. were obtained by absorbing out the subpopulation of antibodies elicited against FeLV proteins RESULTS by use of an immunoaffinity column in which Preparation of antisera directed against detergent-disrupted FeLV was immobilized to a FeSV-coded polyproteins. The known iso- Sepharose 4B matrix. Absorbtion of anti-FeLV lates of FeSV, including the SM, GA, and ST antibodies was monitored by the residual ability strains, code for high-molecular-weight polypro- of the serum to precipitate of '251-labeled FeLV teins that contain the amino-terminal moiety of structural proteins and [35S]methionine-labeled FeLV helper gag gene-coded precursor protein gag and env gene-coded proteins from FeLVand polypeptides of as yet unknown origin (25, infected cells. The properties of these antisera 31, 35). In the present study, we prepared spe- are summarized in Table 1. cific antibodies against these polyproteins by The immune sera, along with monospecific different experimental approaches. Polyproteins antibodies against purified type C viral proteins, coded for by each of the three FeSV strains were including FeLV p15, p30, and gp7O and partially purified from FeSV (WM 292A) pseu- Rauscher-MuLV plO and reverse transcriptase, dotype virions by gel filtration chromatography were tested for their ability to immunoprecipiin the presence of 6 M guanidine-hydrochloride tate [35S]methionine-labeled extracts of MvlLu and utilized as immunogens. In addition, goats and NRK cells nonproductively transformed

TABLE 1. Properties of caprine antisera elicited against the translational products of the SM, GA, and ST strains of FeSV for precipitation of I125 Titers FeLV labeled structural proteins

Antisera

Precipitation of in vivo [3S]methio-

traeslationledFL

productSb Elicited against:

Absorbed with:

Designation

p15

p12

p30

gp7

gag gene env gene | precursor precursor

+ + Partially purified SM-FeSV-coded 1:2,000 1:2,400 T 1:4,000 1:2400d aSMpp, 180,000-dalc polyprotein FeLV aSMpp